Phosphate linked cytosine-guanine (CpG) dinucleotides are statistically underrepresented in the human genome. When they are present, CpG dinucleotides tend to be located within repetitive sequences characterized by low levels of gene expression. Such CpG dinucleotides also tend to feature a methylated cytosine residue.
CpG islands, on the other hand, are genomic sequences with a high density of CpG dinucleotides relative to the rest of the genome. CpG islands include statistical clusters of CpG dinucleotides. While some CpG islands are associated with the promoter region or 5′ end of coding sequences, others are located in introns or genomic regions not known to be associated with coding sequences. CpG islands may be methylated or unmethylated in normal tissues. The methylation pattern of CpG islands may control the expression of tissue specific genes and imprinted genes. Methylation of CpG islands within a gene's promoter regions has been associated with downregulation or silencing of the associated gene. CpG islands may be methylated to varying densities within the same tissue. An increase in methylation of normally unmethylated CpG islands is observed in aging tissues, even as the overall methylcytosine content of the DNA is reduced.
Aberrant methylation patterns are especially notable in cancer cells. For example, hypermethylation has been detected in a number of cancer tissues. Aberrant methylation of cytosines within CpG islands may be a primary epigenetic event that acts to suppress the expression of genes involved in critical cellular processes, such as DNA damage repair, hormone response, cell-cycle control, and tumor-cell adhesion/metastasis, leading to tumor initiation, progression, and metastasis (Li et al., Biochim. Biophys. Acta, 1704: 87-102 (2004)). Aberrant methylation of CpG islands may also be a secondary epigenetic event or a symptom of an upstream abnormality that is the primary event leading to cancer.
It has been proposed that a unique profile of promoter methylation exists for each human cancer, wherein some methylation characteristics are shared and others are cancer-type specific (Esteller et al., Cancer Res., 61: 3225-3229 (2001), U.S. Pat. No. 7,112,404, and U.S. Patent Application Publication Nos. 2005/0153296 and 2005/0164193). Given that aberrant methylation represents new information not normally present in genomic DNA and that aberrant methylation is a common DNA modification and affects a large number of genomic targets, a number of diagnostic and prognostic tests may be developed that assay for the methylation status of one or more target CpGs. Such tests may be based on CpGs that are aberrantly hypermethylated or hypomethylated in diseased tissues. They may also be based on changes in methylation density in CpG islands. Target CpGs can correlate with a risk for cancer, the presence of cancer, or a particular cancer phenotype, such as prognosis or response profile to treatment regimen.
In addition to selecting appropriate target CpGs, the reliability and cost-effectiveness of CpG methylation diagnostic and prognostic tests may also be improved through the use of appropriate controls that provide information about the integrity of the reagents, equipment, and underlying reactions on which the methylation test depends.
Methods for analyzing nucleic acid methylation that include separate, external controls have been described. For example, WO 2007/033834 describes using a parallel, in vitro methylated nucleic acid as a positive control. The positive control is generated by separating an aliquot of genomic DNA and methylating the separated DNA with a methyl transferase, i.e., a methylase, which preferably methylates sequences within specific sequence recognition sites. In this regard, the disclosed method preferably includes using M.SssI methylase, which preferentially methylates cytosines in CpG dinucleotides, to generate positive controls for analyzing CpG methylation in genomic DNA.
Improved controls for use in methylation analysis are highly desirable, for example, in high throughput diagnostic and prognostic tests, where efficiencies related to the smaller reaction numbers, reduced reagent consumption, and increased confidence in test results may be especially valuable. Improved test controls are also highly desirable in diagnostic and prognostic tests for methylation of samples that include small amounts of DNA such as, for example, circulating DNA in bodily fluid samples.